U.S. patent application number 10/468543 was filed with the patent office on 2004-05-13 for lectins for analyzing sugar chains and method of using the same.
Invention is credited to Irimura, Tatsuro, Matsumoto, Mariko, Ono, Takashi, Yim, Mijung.
Application Number | 20040091938 10/468543 |
Document ID | / |
Family ID | 18906248 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040091938 |
Kind Code |
A1 |
Irimura, Tatsuro ; et
al. |
May 13, 2004 |
Lectins for analyzing sugar chains and method of using the same
Abstract
It is expected that lectins of a needed number of types
appropriately corresponding the variation of sugar chains, if
available, are highly useful as tools for analyzing various sugar
chains. From a lectin library containing plural types of lectins or
an arbitrary mass of lectins containing plural types of lectins,
lectins needed for analyzing sugar chains are selected to thereby
construct a lectin sub-library. Using the lectin sub-library thus
constructed, cells having sugar chains are identified. Moreover, an
indication method whereby cells can be conveniently identified is
provided.
Inventors: |
Irimura, Tatsuro; (Tokyo,
JP) ; Matsumoto, Mariko; (Kanagawa, JP) ; Yim,
Mijung; (Seoul, KR) ; Ono, Takashi;
(Yamanashi, JP) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Family ID: |
18906248 |
Appl. No.: |
10/468543 |
Filed: |
December 29, 2003 |
PCT Filed: |
February 20, 2002 |
PCT NO: |
PCT/JP02/01503 |
Current U.S.
Class: |
435/7.1 ;
435/6.16; 506/18; 506/19; 530/370; 530/395; 530/396 |
Current CPC
Class: |
C07K 2319/02 20130101;
C07K 14/42 20130101 |
Class at
Publication: |
435/007.1 ;
530/370; 530/395; 530/396; 435/006 |
International
Class: |
G01N 033/53; C07K
014/415; C07K 014/47; C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2001 |
JP |
2001-44221 |
Claims
1. A lectin sub-library for analyzing sugar chains, comprising a
series of lectins each having a modification at a predetermined
site on the lectin, or plural types of lectins selected by a
panning with cells or pseudo-cells having predetermined sugar
chains.
2. A method of analyzing sugar chains, which comprises using the
lectin sub-library according to claim 1.
3. A tool for diagnosing diseases, comprising the lectin
sub-library according to claim 1.
4. A solid carrier for analyzing sugar chains, wherein different
types of lectins selected for distinguishing sugar chains, from the
lectin sub-library according to claim 1, are immobilized in a
predetermined order.
5. A solid carrier wherein lectin-immobilizing regions are arranged
in a suitable order for distinguishing sugar chains, and the region
contains a lectin selected for analyzing sugar chains.
6. The solid carrier according to claim 5, characterized in that
the selected lectin is obtained by mutating a part of the gene
encoding a lectin.
7. The solid carrier according to claim 6, characterized in that
the lectin to be mutated is MAH lectin.
8. A method of displaying a cell discrimination pattern by using a
solid carrier wherein lectin-immobilizing regions are arranged in a
suitable order for analyzing sugar chains, and the region contains
a lectin selected for distinguishing sugar chains.
9. The method according to claim 8, characterized in that the
selected lectin is obtained by mutating a part of the gene encoding
a lectin.
10. The method according to claim 9, characterized in that the
lectin to be mutated is MAH lectin.
11. A sugar chain-comparing tool and method comprising using at
least one lectin selected from a plurality of lectins known to bind
to at least 2 types of sugar chains, and the selected lectin shows
a greater difference with respect to the ability in binding to one
type of sugar chain relative to another type of sugar chain in said
2 types of sugar chain.
12. A sugar chain-comparing tool and method according to claim 11,
further comprising using at least one lectin showing a smaller
difference with respect to the ability in binding to one type of
sugar chain relative to another type of sugar chain in said 2 types
of sugar chain.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to a technique of analyzing
cell surface sugar chains or the like by using lectins. In
particular, the present invention relates to a sugar chains
analyzing library and sub-library comprising lectins that can
analyze sugar chains, and relates to a method of analyzing sugar
chains by using the library and sub-library for analyzing sugar
chains. Further, the present invention relates to a library and
sub-library of lectins for use in identifying cells, which can
easily and accurately identify the type of particular cells. The
present invention also relates to a method of identifying the type
of cells by using the library and sub-library for identifying
cells.
BACKGROUND ART
[0002] In recent years, it has been said that if the code recorded
in sugar chains could be decoded, new horizons of biology would be
opened. As a tool for analyzing various sugar chains or as a
molecular candidate decoding such a code in the living body,
carbohydrates-binding proteins (lectins) can be mentioned. However,
each individual lectin cannot give macroscopic information for
appropriately reflecting the variety of sugar chains.
[0003] That is, it can duly be expected that if a number of
different types of lectins could be obtained, sufficient enough to
adequately correspond to the variation of sugar chains, they can
constitute a useful tool for analyzing various types of sugar
chains sugar chains.
DISCLOSURE OF INVENTION
[0004] In view of the problem described above, a lectin sub-library
is constructed in the present invention by selecting lectins
necessary for analyzing sugar chains, from a lectin library
comprising plural types of lectins. By using the lectin sub-library
thus constructed, cells having sugar chains are identified.
Further, a method of display capable of easily identifying cells is
provided herein. That is, sugar chains are hardly specified by
using one type of lectin, but they can be identified, by using a
lectin sub-library comprising a combination of different types of
lectins. Further, even in the case that the sugar chains structure
cannot be fully specified in details, the lectin sub-library can
still be used as a tool to identify and to make a comparison of
sugar chains wherein a difference in cells (including a difference
in type and a difference between normality and abnormality)
represented by a difference of sugar chains, or a difference in the
glycosylation modification of proteins can be recognized, or a
substance carrying sugar chains as markers can be discriminated. On
the other hand, when the relationship is clear between a lectin
sub-library and cells, glycosylation modification of glycoproteins,
or sugar chains markers, the difference in cells, the difference of
glycosylation modification of glycoproteins, sugar chains markers
can be identified from the relationship, without elucidating the
actual structure of sugar chains.
[0005] In particular, the present invention provides:
[0006] (1) A lectin sub-library for analyzing sugar chains,
comprising a series of lectins each having a modification at a
predetermined site on the lectin, or plural types of lectins
obtained by a panning with cells or pseudo-cells having
predetermined sugar chains. The panning may be conducted for a
lectin library composed of plural types of lectins. Incidentally,
insofar as the function of a sub-library can still be maintained as
a whole, some elements (lectins) of the sub-library may be omitted.
Similarly, insofar as the function of a library can still be
maintained as a whole, some elements of the library may also be
omitted.
[0007] The present invention further provides:
[0008] (2) A method of analyzing sugar chains, which comprises
using a cell-identifying lectin sub-library comprising a series of
lectins each having a modification in a predetermined site in the
lectin, or plural types of lectins obtained by pannings with cells
or pseudo-cells having sugar chains.
[0009] (3) A tool for diagnosing diseases, which comprises using
the lectin sub-library as described in the above-mentioned (1).
[0010] (4) A solid carrier wherein lectin-immobilizing regions each
of which contains a lectin selected to identify sugar chains, are
arranged in a suitable order for identifying sugar chains. The
lectin-immobilizing region may be a well or a simple spot, e.g. a
region not particularly delimitated by an external structure, or
may be a part or place where lectins can be immobilized. The solid
carrier may be in a form of chip or plate or any other forms of
solid material capable of forming the lectin-immobilizing regions
thereon.
[0011] (5) The solid carrier according to the above-mentioned (4),
characterized in that the selected lectin has been obtained by gene
mutation.
[0012] (6) The solid carrier according to the above-mentioned (5),
characterized in that the predetermined lectin is MAH lectin.
[0013] (7) A method of display to identify cell surface sugar
chains binding pattern by using a solid carrier wherein
lectin-immobilizing regions are arranged in a suitable order for
identifying/analysing sugar chains, and each region contains a
lectin selected for identifying/analysing sugar chains.
[0014] (8) The method of display according to the above-mentioned
(7), characterized in that the selected lectin has been obtained by
gene mutation.
[0015] (9) The method of display according to the above-mentioned
(8), wherein the predetermined lectin is MAH lectin.
[0016] (10) A solid carrier for analyzing/comparing sugar chains,
wherein different types of lectins having specific sugar chains
binding specificities to the cell surface sugar chains are
immobilized in a predetermined order.
[0017] (11) A sugar chains-comparing tool and method comprising
using at least one lectin selected from a plurality of lectins
known to bind to at least 2 types of sugar chains. The selected
lectins show a greater difference with respect to the binding
activity to the 2 types of carbohydrates. When a value "A" that
represents the ability of a certain lectin to bind to one type of
sugar chain, is greater than a value "B" that represents the
ability of the lectin to bind to another type of sugar chain, the
relative difference in the binding ability can be expressed as
(A-B)/(A+B)/2). The greater difference with respect to the binding
capability may refer to those relative differences greater than the
average relative difference in the binding ability of all the
lectins, or when the relative differences are arranged in the order
of from the greatest to smallest differences, the great relative
difference may refer to those relative differences of from the
greatest one to a predetermined one (for example, 1, 2, 3, 4 etc.).
The sugar chains-comparing tool may contain a solid carrier-having
lectins arranged in a suitable order to compare sugar chains.
[0018] (12) The sugar chains-comparing tool and method according to
the above-mentioned (11), further comprising using at least one
lectin showing a smaller difference with respect to the ability in
binding to one type of sugar chains relative to another type of
sugar chains in said 2 types of sugar chains. The small relative
difference may refer to those relative differences smaller than the
average relative difference in the binding ability of all the
lectins, or when the relative differences are arranged in the order
of from the smallest to greatest differences, the small relative
difference may refer to those relative differences of from the
smallest one to a predetermined one (for example, 1, 2, 3, 4
etc.).
[0019] Generally, the term "lectin" is a generic name of proteins
specifically recognizing and binding to sugar chains, which are
roughly divided into animal lectins and plant lectins. Among the
plant lectins, leguminous lectins constitute a large lectin family.
The lectin is composed of a dimer or tetramer of subunits each
having a molecular weight of 30,000. Each subunit has one sugar
chains-recognizing site. Lectin is sometime defined in a narrow
sense as "a multivalent, carbohydrates binding protein excluding
enzymes or antibodies, or defined as a glycoprotein", or can be
defined as "a carbohydrates recognizing protein binding and
cross-linking to carbohydrates" in a broad sense, and has thus the
ability to identify and analyze many types of a carbohydrates
substantially. Particularly, the variation of carbohydrates may
reflect the variation of cells having such carbohydrates on the
surface. Therefore, cells can be identified by
identifying/analyzing their cell surface carbohydrates.
[0020] Unlike antibodies, lectins are found in every organ and
tissue in almost all living things. Further, the structures of
lectins are varied, while the structures of antibodies are
similar.
[0021] In the present invention, leguminous lectins are chosen as
one example of a large number of various lectins to explain the
present invention as completed. However, lectins other than
leguminous lectins can be used in a similar manner, and as a matter
of course, the present invention is not limited to such leguminous
lectins.
[0022] For example, lectins having specific binding properties to
certain sugar chains may be selected and used without any
modification as a lectin library, or mutated lectins at least a
part of which carbohydrates-recognizing regions are genetically
engineered by random mutation can consist a lectin library as
well.
[0023] Specifically, the terms "certain sugar chains" encompass the
cases where sugar chains are defined at least partially, or sugar
chains specified by a process of synthesizing/engineering the
carbohydrates without knowing the exact structure. Further, the
diversity (or an abnormality) in glycosylation of glycoproteins may
result different sugar chains structure and this may also be an
example of the subjects having "certain sugar chains" and
glycoprotein itself can be used to select specific lectins from a
lectin library. Further, cell surface sugar chains are
characteristic and sugar chains are different from cells to cells,
and thus "certain sugar chains" may be read as "certain cells" and
"certain cells" can be used to select specific lectins from a
lectin library. Further, if a cell has different cell surface sugar
chains depending on the environment at the time of glycosylation,
the cell cycle (for example a stage before differentiation or a
differentiated state) may be identified. This is because
glycosylation patterns depends on the cell cycle, environment and
cell surface carbohydrates depends on diseases, aging etc.
[0024] The phrase "lectins having specific binding properties" is
used because not all the mutated lectins have the same binding
specificities, in terms of the binding preference to specific
carbohydrates. When a parental lectin has a specific binding
specificity to a specific carbohydrate structure, there is a high
possibility that the mutated lectins generated from the gene
mutation of its carbohydrates-binding site have a similar
carbohydrate binding specificities pattern. However, mutated
lectins have different binding preference each other and also
different from that of the parental lectin. In this way, a
plurality of lectins would be obtained, each showing different
preference and capability to bind to a certain sugar chain.
[0025] The phrase "at least a part of which
carbohydrates-recognizing regions" is given because lectins
recognize carbohydrates by cross-linking to its
carbohydrate-binding site, a certain region in its whole structure.
The terms "at least a part" is given because gene mutation
corresponding to the whole structure of the lectin is not always
necessary.
[0026] The terms "genetically engineered" is given because chemical
modification of an intact lectin is simply difficult and
inefficient. Therefore, in the method, genes encoding "carbohydrate
recognition regions" are genetically mutated and is transfected
into a suitable host such as Escherichia coli, to produce a mutated
lectin. The term "random mutation" means random genetic mutation,
and also predetermined gene mutations to produce random lectins.
The mutation used herein means the gene mutation by swapping
nucleic acid base without changing the length of the gene or by
making the length of the gene longer or shorter. This also applies
to the mutated lectins produced from the mutations.
[0027] A lectin collection containing a plurality of lectins
obtained in this manner may be called as a lectin library. A
plurality of lectins is contained therein because a single lectin
is not always sufficient for identifying various sugar chains.
[0028] Site-directed mutagenesis is performed because only the
carobohydrate-binding site in the lectin molecule engages in the
recognition of carbohydrates. By modifying such a specific site, a
preferable lectin library would effectively be obtained. Of course,
there may be the case where the effect of the lectin library can be
evaluated from some experimental results, and thus it is not always
necessary for the mutated site to be limited to such a specific
site.
[0029] The method of identifying sugar chains (or a cell having a
certain sugar chains) by a lectin library involves examining the
binding of specific sugar chains to the lectins consisted the
lectin library as described above, and then using the same lectin
library to classify the carbohydrates to be identified, by
evaluation the binding to the lectin library with two ranks, i.e.
the presence of a binding ability (showing bonding) or the absence
of a binding ability (showing no bonding) or with three ranks, i.e.
the two ranks plus an intermediate rank or by multi-stage
evaluation (or analog evaluation) with increased ranks, thereby
distinguishing the type of the sugar chains in terms of the ability
to bind to a plurality of lectins. Further, the method may also
involve the identification/analysis of the binding ability by
analyzing its binding pattern.
[0030] There may be various methods of judging the binding ability,
and for example, the binding ability may be judged by comparison
with a standard sample previously prepared.
[0031] As used herein, the "cells or pseudo-cells having
predetermined sugar chains" have sugar chains, or mimic of the cell
surface sugar chains. Accordingly, when a lectin sub-library is
prepared for cell having carbohydrate A, carbohydrate A or mimic-A
can usually be used to get an appropriate sub-library. The terms
"analyzing sugar chain" may include identification of cells having
sugar chain on the surface, diagnosis of sugar chain-modifidied
serum glycoproteins (glycoform), diagnosis of diseases, and other
analysis, and the "method of analyzing sugar chains" includes
methods for conducting the above. The tool for diagnosing diseases
by using a lectin sub-library for analyzing sugar chain, comprising
a series of lectins prepared by introducing modifications at a
predetermined site on the lectin, or plural types of lectins
selected by panning with cells or pseudo-cells having predetermined
sugar chains refers to a tool (diagnostic reagent, diagnostic
apparatus, etc.) to judge the state of a disease (or health) when
sugar chains on cell surface or glycoproteins are different in the
two states, and a tool to examine the changes of the sugar chains
through the lectin-binding specificities corresponding to the
changes. It is not always necessary to determine the exact
structure (or state) of the sugar chain.
[0032] The phrase "lectin-immobilizing regions are arranged in a
suitable order for identifying/analyzing sugar chains, and the
region contains a lectin selected for identifying/analyzing sugar
chains" has a broad concept encompassing "wells or spots containing
lectins selected for identifying cells, are arranged in a suitable
and appropriate order for the cell identification", and the phrase
"wells or spots containing lectins selected for identifying cells
are arranged in a suitable and appropriate order for the cell
identification" involves arranging wells or spots containing the
selected lectins in predetermined positions so as to make their
visual pattern easily recognizable for the identification, and if a
standard pattern has been established, the phrase involves
arranging the wells or spots in positions corresponding to those in
the standard-pattern. As used herein, "display" may be attained by
either direct or indirect display. The chip can be used to provide
a visual pattern obtained by sugar chain structure information, and
on the basis of this information, the cells can be identified. To
identify cells by enormous pattern information, visual judgment by
the naked eye or any other methods is often insufficient, and thus
the cells may be identified by cluster analysis with a computer. In
the cluster analysis, a computer may be used to examine which part
indicates a significant difference between subjects to be compared
(for example, a standard and an object of examination or a control
and an object of comparison), and general cluster analysis software
can also be used.
[0033] In the phrase "solid carrier for analyzing/comparing sugar
chains wherein different types of lectins are immobilized in a
predetermined order", the terms "immobilized in a predetermined
order" means that the position at which different types of lectin
is immobilized, may be arranged depending on intention of using
sugar chain information for cell analysis or disease diagnosis.
This means, the order to immobilize lectins may be changed
depending on the cell types and diseases. Similarly, in the "chip
wherein wells containing lectins selected for identifying cells are
arranged in a suitable and appropriate order for the cell
identification", the order may be suitably varied depending on the
intention of using sugar chain information for cell analysis or
disease diagnosis. The order may preferably be optimized so as to
be adapted to the intended cells or diseases. The solid carrier may
be in a form of chip or a plate, or any other form of solid
materials that can provide lectin-immobilizing regions. That is,
the "solid carrier for analyzing/comapring sugar chains" includes
"a test plate for analyzing sugar chains". The "test plate for
analyzing sugar chains" includes a test paper using a paper such as
a filter paper, or a glass plate or a substrate made of other
material having predetermined lectins immobilized thereon. Other
material for the substrate may include synthetic resin (including
plastics), metals (including platinum, silver, copper, gold,
silicon, etc.), mica and mixtures thereof. The "test plate for
analyzing sugar chains" can also be used as a diagnostic
reagent.
BRIEF DESCRIPTION OF DRAWINGS
[0034] FIG. 1 shows the introduction of mutation in MAH
carbohydrate recognition domain.
[0035] FIG. 2 is a photograph showing expression of recombinant MAH
mutant lectins in transformed Escherichia coli induced by isopropyl
thiogalactoside.
[0036] FIG. 3 is a list showing the amino acid sequences of the
carbohydrate recognition domains of 16 mutant MAH clones.
[0037] FIG. 4 shows the hemagglutination activity of 16 mutant
lectins (mutant MAH lectins) on erythrocytes from different types
of animals.
[0038] FIG. 5 shows a mutation site in the carbohydrate binding
site of MAH.
[0039] FIG. 6 is a graph showing the ability of wild-type
MAH-expressing phage to bind to anti-MAH antibody.
[0040] FIG. 7 is a graph showing the ability of wild-type
MAH-expressing phage to bind to human glycophorin.
[0041] FIG. 8 is a graph showing the sugar chain specificity of
human erythrocyte-specific clones obtained by panning.
[0042] FIG. 9 shows the results of binding activity of the clones
(1 to 18) to Caco-2 cells measured by a conventional cell-ELISA
method.
[0043] FIG. 10 shows the results of binding activity of the clones
(19 to 35) to Caco-2 cells measured by a conventional cell-ELISA
method.
[0044] FIG. 11 is a graph showing binding patterns of
differentiated or undifferentiated Caco-2 cells to the clones in a
lectin library.
[0045] FIG. 12 are graphs showing binding patterns of various cells
to commercially available lectins.
[0046] FIG. 13 illustrates one example of lectin chips and a method
of using the same (for use in IgA discrimination).
[0047] FIG. 14 illustrates one example of lectin chips and a method
of using the same (for use in profiling of differentiation of
osteoblast).
DETAILED EXPLANATION OF THE INVENTION
[0048] Hereinafter, the present invention is explained in more
detail by reference to working examples.
[0049] At least a part of a region considered to correspond to 6
the carbohydrates-binding site of Maackia amurensis hemagglutinin
(MAH), which is a leguminous lectin specific for sialic
acid-containing carbohydrates, is mutated at random by genetic
engineering techniques, and from the resulting randomly mutated
lectins, an artificial library was prepared capable of identifying
the sugar chains variations in different type of sugar chains.
Further, by using the resulting artificial lectin library, it was
attempted to identify different type of cells based on the binding
pattern to the lectins in the library. Then, from the artificial
lectin library, a screening system established for selecting the
lectins specific for biologically important sugar chains.
[0050] It is known that a vast variety of complex carbohydrates
having slightly different structures, exist among mammalian cells.
Formation of lectins capable of distinguishing such various
structures is very useful.
[0051] Such lectins can be obtained from Maackia amurensis
hemagglutinin (MAH) by genetic engineering techniques.
[0052] MAH can recognize a carbohydrates whose hydrocarbon sequence
comprises a sialic acid residue, e.g.
Neu5Ac.alpha.2-3Gal.beta.1-3(Neu5Ac- .alpha.2-6)GalNAc(4). Another
isolectin engineering Maackia amurensis leukoagglutinin (MAL) can
specifically recognize a sequence
Neu5Ac.alpha.2-3Gal.beta.1-4GlcNAc(5), and both lectins are unique
among other lectins including leguminous lectins.
[0053] MAH is a dimer of subunits having a relative molecular mass
of 29,000.
[0054] A nucleotide sequence of cDNA encoding MAH and an amino acid
sequence deduced therefrom show that MAH consists of 287 amino
acids and has a single peptide consisting of 30 amino acids.
[0055] The putative carbohydrate-recognizing domain of MAH was
identified by comparing its amino acid sequence with amino acid
sequences of other leguminous lectins (7) and by a study of genetic
mutation on this domain defined by amino acids endowing MAH with
binding characteristics.
[0056] Further, those observations were confirmed from a computer
model of a three-dimensional structure of MAH containing
Neu5Ac.alpha.2-3Gal.alpha-
.1-3(Neu5Ac.alpha.2.noteq.6)GalNAc(8).
[0057] FIGS. 1A to C are drawings showing the introduction of a
mutation in MAH carbohydrate-recognition domain. A is an
illustration showing a partial overlap extension method for
introducing a mutation. 1B is a drawing showing the
carbohydrates-recognition domain of MAH, where mutation is
introduced at X sites. 1C is a photograph of agarose gel
electrophoresis showing identification of a DNA of mutant MAH. In
1C, lane 1 is a product AB (up to 400 bp), lane 2 is a product CD
(up to 400 bp), lane 3 is a product AD (up to 800 pb), lane 4 is a
DNA of wild-type MAH (up to 800 pb); M is DNA marker (100 bp DNA
Ladder, Life Technologies, Inc., MD, USA).
[0058] FIG. 2 is a photograph showing expression of recombinant MAH
mutant lectins in transformed Escherichia coli induced by isopropyl
thiogalactoside. 2A is a photograph showing the result of
polyacrylamide gel electrophoresis analysis of recombinant mutant
MAH lectins. 2B is a photograph showing the result of Western
blotting analysis of recombinant mutant MAH lectins with an
anti-MAH polyclonal antibody. In FIG. 2, lane N represents E. coli
carrying pGEX-2T plasmid, lane P represents E. coli carrying
pGEX-2T/wild-type MAH plasmid, and lanes 1 to 16 represent E. coli
carrying pGEX-2T/mutant MAH plasmids (corresponding to clones 1 to
16, respectively). Further, their relative molecular masses are
shown in the left, and the arrow indicates recombinant MAH mutant
lectins.
[0059] FIG. 3 is a list showing the result of deduced amino
sequences of sugar-recognition domains in 16 mutant MAH clones.
[0060] FIG. 4 are graphs showing the result of the hemagglutination
activities of 16 mutant lectins on erythrocytes from different
kinds of animals. The activity of each mutant lectin is shown in
terms of minimum hemagglutination concentration relative to maximum
hemagglutination concentration assumed to be 1. Nos. 1 to 16 refer
to clones 1 to 16, and No. 17 refers to a negative control
(GST).
EXAMPLE 1
Preparation of an Artificial Lectin Library
[0061] On the basis of information obtained from the amino acid
sequences of various leguminous lectins and carbohydrate-binding
sites thereof, a nucleotide sequence encoding the
carbohydrate-binding site at the 127- to 137-positions in 285 amino
acids of MAH lectin was randomized by overlap extension PCR using
synthetic oligonucleotides primers. However, aspartic acid 135
considered to be essential for interaction with sialic acid and
aspartic acid 127 and histidine 132 considered to be essential for
coordination with a metal ion were fixed (FIG. 5). On determining
the DNA sequence by the dye terminator method, it was confirmed
that the obtained mutant MAHs, excepting the initially fixed 3
amino acids, have different amino acid sequences with each other,
indicating that the randomization of MAH lectin has been
attained.
[0062] [Discrimination and Identification of Cells by the Lectin
Library]
[0063] The prepared lectin library was integrated in a vector pGEX
and expressed as GST fusion protein in Escherichia coli. Fifty
clones were selected at random, and from these clones, 16 clones
considered to express the protein were obtained. Western blotting
of the proteins with an anti-MAH polyclonal antibody indicated that
all the clones remained reactive. After the determination of the
DNA sequences of these clones, it was confirmed that the amino acid
sequences of the sugar-binding sites in all clones, excepting the
fixed amino acids, had been changed.
[0064] By utilizing a difference in sugar-binding specificity among
the resulting 16 clones, it was attempted to distinguish and
identify erythrocytes of different kinds of animals, on which sugar
chains comprising a sialic acid but having different structure is
considered to exist. FIG. 4 shows the result of the experiments
where the agglutination titer of each lectin of the 16 clones is
compared. In the graphs, the data were standardized by taking the
maximum agglutination by the most reactive lectin for the
respective erythrocytes as "1" (full-scale). The respective
erythrocytes were found to show inherent characteristics in terms
of the relative agglutination titer against the 16 mutant
lectins.
[0065] This experiment suggests that the difference in the type of
cell can be distinguished by referring to the binding patterns with
respect to a plurality of lectins.
[0066] [Preparation of a Phage Library]
[0067] The artificial lectin library would be further developed by
selecting lectins specific for a biologically useful carbohydrate
chain. For this selection, a system capable of expression cloning
and dealing with a large number of libraries should be used. In
this context, a phage display system was introduced. It was
attempted to use an expression vector .lambda.foo and then a T7
phage display system. However, the growth of lectin-expressing
phage was delayed as compared with the counterpart phage not
carrying the gene, and it was found difficult to concentrate the
desired phage by panning. (However, this does not necessarily mean
that the expression vector .lambda.foo or the T7 phage display
system is excluded from the present invention.)
[0068] Incidentally, panning refers to a technique of recovering
phage by utilizing the ability of a protein expressed on the phage
to bind to the binding partner (for example, the binding ability
between antigen and antibody, lectin and carbohydrate, receptor and
ligand, etc.) For example, a substance that binds to the protein
expressed on phage is immobilized on a plate, then a phage solution
is added to the plate thereby allowing the expressed proteins,
phage and other materials to bind to the said substance, then the
phage solution is discharged, and the expressed proteins, phage and
other materials thus binding to the substance are recovered. The
recovered phage can be used to infect with Escherichia coli to
proliferate and then subjected again to (the above-described)
panning to recover highly binding phage.
[0069] Then, it was attempted to use phageimids pComb3 and pComb8.
It was first examined whether a lectin expressed on the surface of
both the phages had an activity or not by expressing wild-type MAH.
When the phages considered to express wild-type MAH were examined
for their reactivity with an anti-MAH antibody, both phages were
bound specifically to the anti-MAH antibody. pComb8 expressing a
plurality of MAH proteins showed a stronger binding ability than
pComb3 expressing a single MAH protein (FIG. 6).
[0070] It was examined whether the wild-type MAH expressed on the
phages was maintaining the carbohydrate-binding activity by
measuring the ability to agglutinate erythrocytes. Both the phages
considered to express wild-type MAH agglutinated untreated
erythrocytes, but did not agglutinate erythrocytes treated with
siliadase. The minimum titer of both the phages necessary for
hemagglutination was about 1.times.10.sup.10 cfu. Thus, it was
revealed that the activity of MAH can be maintained on the phage,
and therefore MAH having a carbohydrate binding activity can be
expressed on the pComb phage.
[0071] The activities of both phages were further examined in more
detail in terms of the ability to bind to human glycophorin (sialic
acid-containing glycoprotein). It was unexpectedly found that
pComb3 expressing a single MAH protein per phage had a higher
activity (FIG. 7). The minimum titer of pComb3 phage detectable by
ELISA in terms of the ability to bind to human glycophorin was
about 1.times.10.sup.7 cfu.
[0072] [Selection of Specific Lectins from a Phage Library]
[0073] On the basis of the above results, the pComb3 phageimid was
used to prepare a lectin library having mutated primary structures.
For preparation of the library in a feasible range, 3 amino acids
that were considered to be essential for interaction with sialic
acid and for coordination with a metal ion, were fixed. Other two
amino acids that are highly conserved among other leguminous
lectins, were also fixed. The remaining 6 amino acids were mutated
at random in the amino acid sequence of the MAH sugar-binding site.
As a result, a library of about 1.times.10.sup.8 lectins considered
to cover the theoretically estimated number of lectins of
6.25.times.10.sup.7 could be prepared.
[0074] For establishing a panning system for the lectin library of
pComb3 phage, a panning with human erythrocytes that strongly bind
to the wild-type MAH was performed. The recovery in the third
panning was about 10 times as high as that in the first panning,
thus revealing that the phage having an ability to bind to human
erythrocytes was concentrated. From the phages obtained after the
third panning, several clones that specifically agglutinate human
erythrocytes but shows different carbohydrates specificity were
obtained (FIG. 8). In this way, a screening system could be
established for selecting lectins specific for biologically
important carbohydrates from a library of lectins having mutated
primary structures.
[0075] [Mutagenesis of the Sugar Chain Recognition Site of MAH]
[0076] The carbohydrates recognition site of MAH was randomized by
site-directed mutagenesis (9, 10) utilizing overlap extension
procedure. In the overlap extension, the following 4 primers were
used.
1TABLE 1 Four primers Primer a: 5'-ccccggatccacatcagatgagctttct-3'
(bp 89-105) Primer b: 5'-atgtcgataatttggatctnnmnnatcmnnmnnatgmnnmn
(bp 457-519) nmnnmnngtcaaactctacagc-3' Primer c:
5'-gatccaaattatcgacatatcggaattgat-3' (bp 502-531). Primer d:
5'-cccgaattcagatcatgcagtgtaacg-3' (bp 847-531) (N represents A, T,
G or C, and M represents A or C)
[0077] MAH cDNA fragment was PCR amplified by using primer pairs of
a and b or c and d with AmpliTaq Gold DNA polymerase (Perkin
Elmer). Standard PCR conditions were used. Specifically, the
reaction mixture was incubated at 95.degree. C. for 9 minutes and
at 94.degree. C. for 1 minute and then subjected to 30 cycles each
consisting of incubation at 94.degree. C. for 1 minute, at
54.degree. C. for 1 minute and at 72.degree. C. for 1 minute
followed by one cycle of incubation at 72.degree. C. for 10
minutes, whereby all amplified fragments were extended certainly to
the 3' end. The 3'-end of the PCR product was polished with a pfu
DNA polymerase (Stratagene La Jolla, Calif.). The PCR product was
purified and quantified by preparative agarose gel electrophoresis.
Equimolar amounts of the two PCR fragments were bound to each other
by 7 cycles each consisting of incubation at 94.degree. C. for 1
minute and at 63.degree. C. for 4 minutes. The product was used as
template DNA in second reaction using primers a and d, whereby a
full-length mutant MAH cDNA was prepared.
[0078] [Expression and Purification of GST Fusion Protein]
[0079] The PCR product containing the full-length mutant MAH cDNA
was digested with BamHI and EcoRI and then ligated to pGEX-2T
previously digested with BamHI and EcoRI. E. coli BL21 strain
containing the expression plasmid was cultured in a
2.times.yeast-trypton medium supplemented with 50 .mu.g/ml
ampicillin, and grown at 37.degree. C. until the absorbance at 660
nm reached 0.8. Expression of GST fusion protein was induced by
adding isopropyl .beta.-D-thiogalactoside (final concentration: 1
mM) and then the cells were incubated at 37.degree. C. for 3 to 4
hours. A pellet of the microbial cells was suspended in Tris
buffered physiological saline (TBS), and sonicated. The insoluble
fraction was washed twice with TBS and dissolved in 8 M urea in
TBS. Thus solubilized inclusion body was refolded by 50-fold
dilution with TBS/1 mM MnCl.sub.2/1 mM CaCl.sub.2. The supernatant
was applied onto a glutathione-Sepharose 4B column (Amersham
Pharmacia Biotech UK Ltd., England), and the GST fusion protein was
purified according to the manufacturer's instructions.
[0080] [Detection of GST Protein by Western 1Blotting]
[0081] The microbial lysate (0.5 .mu.g protein/ml) was separated by
sodium dodecylsulfate-polyacrylamide gel electrophoresis,
transferred onto a nitrocellulose membrane, screened by polyclonal
anti-MAH antibody and then incubated with an alkali
phosphatase-bound anti-rabbit IgG antibody (ZYMED, San Francisco,
Calif.). According to manufacture's instructions, the bound
antibody was visualized with an alkali phosphatase substrate kit
SK-5200 (Vector. Laboratories, Beringam [phonetic], Calif.).
[0082] [Determination of DNA Sequences of Recombinant MAH
Mutants]
[0083] DNA sequences of 16 clones selected at random were
determined by using a sense primer 5'-ttcttgcaccacctgattctc-3' and
an antisense primer 5'-ccgccgttaatccaatcccat-3' with ABI PRISMT.TM.
Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied
Biosystems, California, US).
[0084] [Preparation of Erythrocytes]
[0085] Human venous blood of the O type was taken from a blood
donor by using a previously heparin-treated hypodermic syringe. The
heparin-treated blood was transferred into a glass cylinder and
left until the erythrocytes were precipitated by gravity. After
leukocyte-rich serum and buffy coat were removed, the erythrocyte
layer was washed 3 times by centrifugation with phosphate buffered
saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 1.8 mM
KHPO.sub.4 (pH 7.3)), and the outermost layer was removed carefully
each time of washing after centrifugation. It was confirmed that
the erythrocytes obtained in this manner did not contain leukocytes
or cell debris. Bovine, equine, porcine and chicken erythrocytes
were prepared in the same manner.
[0086] [Hemagglutination Assay]
[0087] A recombinant protein purified from each of the 16 mutant
lectins was diluted twice and put to a U-bottomed plate (Nunc,
N.Y., USA), and to each dilution (0.05 ml) was added an equal
volume of 2% erythrocyte suspension. The mixture was left for 1
hour at room temperature and then examined for
hemagglutination.
[0088] The following gives more specific disclosure.
[0089] [Mutagenesis of the Sugar Chain Recognition Site of MAH]
[0090] The overlap extension method (9, 10) was used to introduce a
mutation into the sugar chain recognition site of MAH. The scheme
(strategy) of this method is shown in FIG. 1.
[0091] Using MAH cDNA cloned in pGEX-2T as a template for PCR
amplification, two different DNA fragments (AB and CD) were formed.
The fragment AB was amplified with primers a and b. Because Asp
135, Asp 127 and His 132 were considered to participate in binding
of MAH to sialic acid, primer b was designed such that while Asp
127, His 132 and Asp 135 resides were stored, the sequence of the
sugar chain recognition site of MAH was mutated (FIG. 1B).
[0092] The fragment CD was amplified with primers c and d. The 5'
end of the primer c had a 12-bp sequence found in the 3' end of the
fragment AB. The fragments AB and CD can be ligated to each other
via this common 12-bp sequence by the overlap extension method to
form an AD product harboring the mutant MAH cDNA. The length (up to
800 bp) of the mutant MAH cDNA was confirmed by agarose gel
electrophoresis (FIG. 1C).
[0093] [Expression of MAH Mutant Lectins and Elucidation of
Characteristics Thereof]
[0094] The mutant MAH cDNA was inserted into a BamHI-EcoRI site in
E. coli expression vector pGEX-2T containing a
glutathione-S-transferase (GST) gene upstream of the insertion
site. GST/mutant lectin fusion proteins were expressed by using
Escherichia coli BL21 as a host strain. The ability of 50 randomly
selected clones to express the recombinant mutant lectins was
examined by polyacrylamide gel electrophoresis. GST/mutant lectin
fusion proteins from 16 clones were electrophoresed to a position
corresponding to an apparent molecular weight of 55,600, which is
predicted with an molecular weight of the GST/mutant lectin fusion
product (FIG. 1A).
[0095] Plasmid DNAs from the 16 clones were sequenced by a dye
deoxy chain terminator method (11). FIG. 3 shows the elucidated
amino acid sequences of the sugar chain recognition sites of these
clones. As expected, the sugar chain recognition sites of the 16
clones were different in amino acid sequence excepting Asp 127, His
132 and Asp 135 residues. Regardless of this difference, all the 16
mutant lectins were detected by Western blotting with an antibody
specific for MAH (FIG. 1B).
[0096] The 16 mutant lectin fusion proteins were purified by
affinity chromatography on a glutathione-Sepharose column. The
concentration was determined by a BCA protein assay kit with bovine
serum albumin as the standard. Profiles of the purified fusion
proteins after polyacrylamide gel electrophoresis in the presence
of SDS suggest that all fusion proteins have a molecular weight of
55,600. These proteins were used in hemagglutination assays.
[0097] [Hemagglutination Activity of the Mutant Lectins]
[0098] Hemagglutination assays using human, bovine, equine, porcine
and chicken erythrocytes revealed that there was a significant
difference in the sugar chain specificity among the 16 mutant
lectins and the wild-type MAH (FIG. 1). Sugar chains on the
surfaces of these erythrocytes are different in the structure,
amount and expression pattern as is evidently shown by comparing
the respective recognition patterns by the mutant lectins (FIG. 4).
Each type of erythrocyte can be easily identified by using these
lectins. In
[0099] In a radial graph in FIG. 4 similar to a form of bicycle
spokes, the 16 different lectins and blank are expressed as
straight lines of the same length, and these straight lines are
arranged radially from one point at equal angles through
360.degree.. In FIG. 4, one radial graph is prepared for
erythrocytes from one type of animal, and this graph shows the
discrimination (or characteristics) of the erythrocytes by the
respective lectins in a visual manner. That is, the maximum
agglutination titer observed is expressed in full scale "1" and
plotted in the outermost side in this graph. The agglutination
titers of the other-lectins are plotted in a similar manner,
respectively, and adjacent plots are linked via a straight line. By
expressing plots in this manner, the positions of the plots is more
easily recognizable than by simple plots, and the resulting graph
in a web form permits discrimination (or characteristics) of the
erythrocytes by the respective lectins to be easily and visually
recognized. The 16 lectins can be arranged so that the similar ones
are placed as adjacent as possible, contrastive ones are placed
apart at 180.degree., and so on. It is preferable that the
arrangement of the plot of lectins is suitably coordinated
depending on the sugar chain to be analyzed. The graph in FIG. 4
shows all the 16 lectins, but the graph may be modified to show 15
or less lectins, wherein some lectins not necessary for
discrimination are omitted, or it may of course include 17 or more
lectins adding thereto some additional lectins. Further selection
of such lectins can be carried out, for example, from the viewpoint
of their ability to bind to intended sugar chains. Referring to
FIG. 4, clone 15 can be selected for distinguishing bovine
erythrocytes from human erythrocytes. This is because clone 15 has
an ability to bind strongly to human erythrocytes but is poor in an
ability to bind to bovine erythrocytes and the relative difference
therebetween is great. Further, clone 13 may also be selected. This
is because clone 13 has the same ability to bind to human
erythrocytes and bovine erythrocytes and the relative difference
therebetween is small. By selecting lectins showing great and small
relative differences respectively for intended erythrocytes,
unknown samples of erythrocytes could be judged easily and
reliably.
[0100] The life cycle of cells in multi-cellular living things is
regulated by stimuli from extracellular environments, and such
stimuli are mediated at least partially via sugar chain and its
recognizing molecules. It has been considered that, as in the case
of receptors specific for antigens in an adapted immune system,
cell surface carbohydrates have a wider variety. However, there is
a limit to the variation of natural lectins capable of recognizing
such tremendous variety of carbohydrates/oligosaccharides.
[0101] In the present invention, a method is provided that enables
the preparation of a number of novel lectins (novel lectin library)
having various specificities. The 16 mutant MAH lectins were
produced. Although their characteristics in terms of carbohydrate
binding specificity has not fully been revealed, it is noteworthy
that the mutants can agglutinate human and animal erythrocytes with
highly specific patterns. The specificity of lectins to distinguish
different animal erythrocytes from one another has not been
achieved except by using specific antibody thereagainst. The
present invention is to prove that a panel of genetically
engineered lectins having various specificities is useful for
distinguishing the type of different cells.
[0102] In the last several years, DNA arrays techniques were
established as a new technology for distinguishing the type of
difference cells thorough profiling of gene expression (15).
However, the phenotype of cells cannot fully be determined only on
the basis of gene expression. Posttranslational modification by
adding sugar chains and control of protein localization are
considered to have essential roles in determining the actual stage
in the differentiation of certain type of cells. Accordingly,
different cells should be distinguished by describing the state of
glucan on the surfaces of the cells. Use of a panel of lectins
having various specificities is the most convenient and effective
means for achievement thereof. Development of a novel lectin
library can significantly expand the applicability of lectins to
ultimately make the identification of cells accurate and reliable.
Although the expression of carbohydrates are very low, and their
structure is complicated, the lectin library and/or lectin
sub-library according to the present invention can be used to
provide a convenient discrimination (or identification) method that
can also be used in clinical fields.
EXAMPLE 2
[Cell Discrimination and Identification Targeted at Caco-2
Cells]
[0103] A mutant MAH lectin library consisting of loop D mutated MAH
lectins was subjected to panning with differentiated Caco-2 cells
as standard cells, and clones used as probes for cell
discrimination and identification of the target Caco-2 cells were
recovered. After the second panning, the concentration of the
binding clones was considered insufficient, and thus in the third
panning, the cells were fixed with glutaraldehyde, and the reaction
time in the panning procedure was changed into o/n.
[0104] [Determination of DNA Sequences of Recovered Clones]
[0105] After the third panning, 64 clones were selected at random
and analyzed for their sequence. The 64 clones consist of 35 clones
having the intended mutations, 7 clones having a stop codon, 17
clones undergoing frame-shift, and 5 clones that could not be
analyzed. Among 35 clones having the intended mutations, there were
no clones having the same sequence as that of the wild type and he
clones have no common sequence at the mutated site (Table 2).
2TABLE 2 Sequences of Clones 3rd clone analysis 1 2 3 4 5
[0106] A large number of clones different from the wild type has
been obtained, these 35 clones can be used as probes for
discrimination and identification of cells. On the other hand,
since no common sequence was detected among these clones, and
clones having a stop codon or undergoing frame-shift were
recovered, the concentrating efficiency in the panning procedure is
considered as not so high in this experiment.
[0107] [Measurement of the Activity of the Recovered Clones]
[0108] Using a lysate of each clone, the activity thereof to bind
to Caco-2 cells was measured by a conventional cell-ELISA method
(FIGS. 9 and 10) All of the 35 clones had an activity to bind to
both differentiated and undifferentiated Caco-2 cells. When binding
patterns of the respective clones were plotted with respect to the
differentiated and undifferentiated Caco-2 cells, a binding pattern
appeared to be unique to Caco-2 cells was obtained and there was a
slight difference in the pattern between the differentiated and
undifferentiated cells (FIG. 11). These results indicate that a
difference in the pattern between the differentiated and
undifferentiated cells can be observed even in the same kind of
cells. This type of graph in FIG. 11 is superior in judging a
slight difference because the results of both the differentiated
and undifferentiated cells can be collectively shown in one
graph.
EXAMPLE 3
[0109] Profiling of various cells was conducted as well. The
commercially available purified lectins lectins (PNA, ABA, VVA-B4,
SNA, UEA-I, WGA, PHA-L4) were immobilized directly onto wells and
sample cells were added to the wells to detect the binding thereof
(reverse cell-ELISA method) (FIG. 12). As can be seen from the
graphs in FIG. 12, a binding pattern unique to each kind of cell is
obtained and expressed in a graph in a radial form similar to a
form of bicycle spokes, whereby characteristics of the cell can be
grasped. The manner of plotting the graph in FIG. 12 is the same as
that in FIGS. 4 and 11 described above.
[0110] [Discrimination of IgA Glycoforms]
[0111] An application of the lectin library to a method of
distinguishing IgA glycoforms is described below.
[0112] 1) A genetically mutated lectin library is prepared by
mutating an amino acid sequence of the sugar chain recognition site
of MAH lectin (Maackia amurensis hemagglutinin lectin)
[0113] 2) Lectins having higher affinity for an IgA from a patient
having IgA nephropathy are selected by the panning method, to
prepare a lectin sub-library.
[0114] 3) Lectins from the lectin sub0 library obtained in step 2)
above, are immobilized on a microtiter plate. Optionally, lectins
sufficiently reflecting a difference between the IgA from the
patients having IgA nephropathy and IgA from healthy persons may be
specifically selected from the lectin sub-library obtained in step
2) above, to prepare the plate.
[0115] 4) Discrimination by a lectin library of IgA glycoforms:
Binding patterns, on the lectin plate, of serum IgA from the
patients having IgA nephropathy and serum IgA from healthy persons
are compared and analyzed.
[0116] 5) Investigation of assay conditions for serum analysis:
Sugar chains are added enzymatically or chemically to IgA from
healthy persons, to prepare artificial IgA. The artificial IgA is
mixed with serum from healthy persons and subjected to pattern
analysis by using the lectin plate. The influence of other serum
proteins present in serum may be examined to optimize assay
conditions.
[0117] IgA nephropathy is a disease in which IgA is precipitated in
renal glomerulus, and the IgA molecule is different in the sugar
chain structure on its hinge region between the IgAs from patients
and healthy persons. The analysis of the sugar chain structure is
conventionally carried out by purifying serum IgA, separating the
hinge-containing peptide fragment by a treatment with trypsin, and
measuring its mass by a mass spectrometer. The hinge region refers
to a region between two H-chain constant domains constituting an
immunoglobulin molecule, and it is assumed that in patients with
IgA nephropathy, there is an abnormality in an O-linked sugar chain
on the hinge region in IgA. Further, an O-linked sugar chain
having, as a fundamental skeleton, a structure composed of N-acetyl
galactosamine (GalNAc), galactose (Gal) and outermost sialic acid,
is also referred to as a mucin type sugar chain. The O-linked type
sugar chain can significantly influence on such as intercellular
interactions, cellular infiltration, adhesion etc.
[0118] The detailed mechanism of the method is as follows: The
O-linked type sugar chains can be attached onto serine and
threonine residues at 5 distinctive sites in the amino acid
sequence of the hinge region in IgA, where patients with IgA
nephropathy have an abnormality in the O-linked sugar chain. There
are 6 different patterns of the O-linked sugar chains, and thus
6.sup.5 (i.e. 7,776) different sugar chain structures can
theoretically occur in relation to the attaching positions in the
hinge region. It is difficult to examine every possibility by a
complicated sugar chain analysis conducted conventionally. However,
the pattern analysis would be feasible by observing ON/OFF signals
toward lectins that recognize the O-linked sugar chain. By
examining ON/OFF toward one lectin, two sugar chain linkage modes
can be determined, and with "n" lectins given, 2.sup.n patterns
would be recognizable. Accordingly, the number of lectins necessary
for analyzing 7,776 sugar chain abnormalities in the hinge is
theoretically 13. This theoretical number is elucidated based on an
assumption that there are lectins capable of clearly reflecting the
structural and positional information of the sugar chain. However,
if information from several hundred lectins is available and the
lectins can be blotted on a substrate, there is a high possibility
that the structural and positional information of the sugar chain
in the IgA hinge can be obtained. To obtain a useful lectin
library, a group of lectins that clearly reflects the difference in
between serum IgAs from patients and from healthy persons, can be
identified by a cluster analysis to construct an effective lectin
sub-library. Using the lectin library, a difference in glycoform
between serum IgA from patients with IgA nephropathy and serum IgA
from healthy persons is analyzed by the pattern information (FIG.
13). FIG. 13 shows that the profiling of IgA1, a glycoprotein
containing a plurality of O-glucans, can easily be carried out.
There is applicability not only to analysis of IgA from patients
with severe diseases such as those in chronic renal insufficiency
but also to early diagnosis of potential patients with a slight
symptom not diagnosed as IgA nephropathy.
[0119] [Method of Distinguishing Osteoblast Subgroup]
[0120] One example of the application of the lectin library to a
method of distinguishing osteoblasts or to a method of detecting a
subgroup different in the stage of differentiation is described
below.
[0121] 1) A genetically mutated lectin library is prepared by
mutating an amino acid sequence of the sugar recognition site of
MAH lectin (Maackia amurensis hemagglutinin lectin).
[0122] 2) Lectins having high affinity for the prepared osteoblasts
are selected from the lectin library by panning to prepare a lectin
sub-library.
[0123] 3) Lectins from the lectin sub-library obtained in the step
2) above, are immobilized on a microtiter plate. Optionally,
lectins sufficiently reflecting the stages of differentiation are
selected from the lectin sub-library obtained in the step 2) above,
to prepare the lectin plate.
[0124] 4) Introduction of the differentiation of osteoblasts and
discrimination by the lectin library: Mesenchymal stem cells are
cultured and separated. Specifically, the mesenchyme stem cells are
cultured and then differentiated into osteoblasts, and the cells on
the 5th day, 10th day, 15th day and 20th day after the initiation
of differentiation are separated.
[0125] 5) The cells separated at each stage in the differentiation
process are analyzed with the lectin plate. The correlation between
the sugar chain structure on the surface of the cell and the
ability to form bone is examined. The ability to form bone is
determined by measuring the activity of bone-type alkali
phosphatase and simultaneously measuring the content of osteocalcin
(preparation of a standard). That is, the separated cells in the
form of a complex with a b-TCP block serving as an anchor for the
osteoblasts are transplanted subcutaneously in the back of a rat,
and 4 weeks and 8 weeks after transplantation, the cells are
removed and examined for (i) osteocalcin content and (ii) bone-type
alkali phosphatase activity.
[0126] 6) Cells whose differentiation process is not
identified/unrevealed are analyzed with the lectin plate and
compared with the standard pattern (see FIG. 14). FIG. 14 shows
that profiling of differentiation of osteoblasts can be easily
conducted.
[0127] As used herein, the mesenchymal stem cells are stem cells
which among those differentiated into tissues or organs, occur in
the bone marrow, and it is confirmed that the mesenchymal stem
cells are differentiated into cells in bone, cartilage, fat, heart,
nerves, liver etc., and have a potential similar to that of
embryonic stem cells (ES cells) which can be differentiated into
almost all tissues. The mesenchymal stem cells can be used as an
anchor for culturing an artificial material based on calcium
phosphate called b-TCP (b-tricalcium phosphate) excellent in
affinity, absorptivity, and bone conductivity in the living
body.
[0128] According to the present invention, the lectin library was
found to be useful for discrimination and identification of
cells.
[0129] By analyzing cell surface sugar chain by this new method,
identification of cells, which has solely been depended on gene
expression only, or antibodies whose antigen recognition is
limited, be easily carried out, and thus this method would be
useful for development of cell transplantation and cell therapy.
Further, this study succeeded in expressing an artificial lectin
library on the surface of phageimid-type phage. Because the method
of selecting lectins having different specificities from a mutant
lectin library was established, it is expected that this system can
be used for obtaining lectins having novel sugar chain specificity
absent in existing lectins or monoclonal antibodies.
[0130] Further, the lectin library of the present invention can be
used to provide a tool for detection of IgA glycoforms or for
discrimination/identification of an osteoblast subgroup. That is,
the lectin library can provide not only a diagnostic reagent for
easily and rapidly analyzing glycoforms of various serum proteins
including IgA, but also a standard design tool for guaranteeing
qualities of cells necessary for bringing regenerative medical
treatment or cellular medical treatment into a practical stage. For
example, these tools can be applied to early discovery of diseases,
to accurate understanding of a morbid state and to
therapeutic/prophylactic medicines, for example by analyzing
glycoforms of immunoglobulins in rheumatism and autoimmune
diseases, glycoforms of specific hormones in patients with cancers
(that is, change in sugar chains of chorionic gonadotropin in
ovarian cancer) and glycoforms of proteins (change in sugar chains
of alpha-fetoprotein at a stage of from hepatitis to hepatoma).
[0131] Because an antibody is characterized by being produced
depending on a difference in species, an antibody specific for
fibroblasts is hardly produced. Therefore, fibroblasts derived from
bone marrow do not have a clear so-called cell surface marker, and
there is no method of directly assaying osteoblasts. Even at
present time, bioassays with animals or indirect bioassays
reflecting the activity of osteoblasts are employed. In such a
field, a lectin chip using the lectin library of the present
invention would be feasible in the discrimination/identification of
osteoblasts, particularly in the discrimination/identification of
an osteoblast subgroup at different stages. By the same approach,
it can duly be expected that the lectin library of the invention
can be applied to the analysis of the dendritic cells derived from
bone marrow and the vascular endothelial cells derived from bone
marrow, and therefore can be used as a standard tool for securing
qualities of cells used in regenerative medical treatment.
[0132] A network for remote diagnosis of cancers at present is
based on pharmacological diagnosis, but it is examined to add a
genetic information thereto. On finding the considerable
involvement of the O-linked sugar chain in the intercellular
interactions, cell infiltration, adhesion etc., it would be
possible to achieve more detailed remote diagnosis of cancers.
Sequence CWU 1
1
95 1 28 DNA Artificial An artificially synthesized primer sequence.
1 ccccggatcc acatcagatg agctttct 28 2 63 DNA Artificial An
artificially synthesized primer sequence. 2 atgtcgataa tttggatctn
nmnnatcmnn mnnatgmnnm nnmnnmnngt caaactctac 60 agc 63 3 30 DNA
Artificial An artificially synthesized primer sequence. 3
gatccaaatt atcgacatat cggaattgat 30 4 27 DNA Artificial An
artificially synthesized primer sequence. 4 cccgaattca gatcatgcag
tgtaacg 27 5 21 DNA Artificial An artificially synthesized primer
sequence. 5 ttcttgcacc acctgattct c 21 6 21 DNA Artificial An
artificially synthesized primer sequence. 6 ccgccgttaa tccaatccca t
21 7 11 PRT Maackia amurensis 7 Asp Thr Tyr Phe Gly His Ser Tyr Asp
Pro Trp 1 5 10 8 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 8 Asp Asn Leu Ile Tyr His Ile Asn Asp
Met Ala 1 5 10 9 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 9 Asp Arg Pro Leu Thr His Ser Asp Asp
Pro Val 1 5 10 10 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 10 Asp Ala Asn Val Leu His Lys Leu Asp
Arg Arg 1 5 10 11 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 11 Asp Val Pro Asn Val His Lys Ile Asp
Tyr Arg 1 5 10 12 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 12 Asp Glu Ser His Cys His Val Glu Asp
Val Glu 1 5 10 13 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 13 Asp Val Met Pro Leu His Leu Gln Asp
Thr Thr 1 5 10 14 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 14 Asp Thr Ser Leu Pro His Val Val Asp
Val Gly 1 5 10 15 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 15 Asp Asn Leu Thr Gly His Leu Pro Asp
Ser Gly 1 5 10 16 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 16 Asp Met His Val Val His Asn Ser Asp
Asn Leu 1 5 10 17 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 17 Asp Leu His Ser Asn His Thr Leu Asp
Lys Gln 1 5 10 18 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 18 Asp Pro Lys Pro Leu His Ala Ile Asp
Ser Ser 1 5 10 19 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 19 Asp Val Ser His Trp His Thr Gln Asp
Tyr Arg 1 5 10 20 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 20 Asp Thr Glu Leu Leu His Thr Asn Asp
His Lys 1 5 10 21 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 21 Asp Ser Ala Ser Asn His Val Ala Asp
Glu Arg 1 5 10 22 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 22 Asp Ser Ser Gly Ile His Asp Leu Asp
Gly Arg 1 5 10 23 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 23 Asp Pro Pro Met Ala His Tyr Arg Asp
Trp Pro 1 5 10 24 33 DNA Maackia amurensis CDS (1)..(33) 24 gac act
tac ttc ggc cat agt tat gat ccc tgg 33 Asp Thr Tyr Phe Gly His Ser
Tyr Asp Pro Trp 1 5 10 25 11 PRT Maackia amurensis 25 Asp Thr Tyr
Phe Gly His Ser Tyr Asp Pro Trp 1 5 10 26 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 26 gac act tac
cag gcg cat att acg gat cat tcg 33 Asp Thr Tyr Gln Ala His Ile Thr
Asp His Ser 1 5 10 27 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 27 Asp Thr Tyr Gln Ala His Ile Thr Asp
His Ser 1 5 10 28 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 28 gac act tac ttg cct cat tcg ttg gat
aat cgt 33 Asp Thr Tyr Leu Pro His Ser Leu Asp Asn Arg 1 5 10 29 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
29 Asp Thr Tyr Leu Pro His Ser Leu Asp Asn Arg 1 5 10 30 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 30
gac act tac gag cgt cat act ggg gat ggt atg 33 Asp Thr Tyr Glu Arg
His Thr Gly Asp Gly Met 1 5 10 31 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 31 Asp Thr Tyr Glu Arg His
Thr Gly Asp Gly Met 1 5 10 32 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 32 gac act tac tcg gct cat
cct ggt gat gtg gtg 33 Asp Thr Tyr Ser Ala His Pro Gly Asp Val Val
1 5 10 33 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 33 Asp Thr Tyr Ser Ala His Pro Gly Asp Val Val 1 5 10
34 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 34 gac act tac gct ctt cat ccg ggg gat tct ttt 33 Asp Thr
Tyr Ala Leu His Pro Gly Asp Ser Phe 1 5 10 35 11 PRT Artificial
Generated from randomly recombinant DNA part of MAH. 35 Asp Thr Tyr
Ala Leu His Pro Gly Asp Ser Phe 1 5 10 36 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 36 gac act tac
tct cgg cat cct ttt gat agt gag 33 Asp Thr Tyr Ser Arg His Pro Phe
Asp Ser Glu 1 5 10 37 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 37 Asp Thr Tyr Ser Arg His Pro Phe Asp
Ser Glu 1 5 10 38 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 38 gac act tac ctt tgt cat tct ttg gat
tgg ctg 33 Asp Thr Tyr Leu Cys His Ser Leu Asp Trp Leu 1 5 10 39 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
39 Asp Thr Tyr Leu Cys His Ser Leu Asp Trp Leu 1 5 10 40 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 40
gac act tac ggg ggg cat tcg cct gat ttt gtt 33 Asp Thr Tyr Gly Gly
His Ser Pro Asp Phe Val 1 5 10 41 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 41 Asp Thr Tyr Gly Gly His
Ser Pro Asp Phe Val 1 5 10 42 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 42 gac act tac tcg gcg cat
tct tcg gat gat gct 33 Asp Thr Tyr Ser Ala His Ser Ser Asp Asp Ala
1 5 10 43 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 43 Asp Thr Tyr Ser Ala His Ser Ser Asp Asp Ala 1 5 10
44 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 44 gac act tac agg gtt cat gcg tct gat gcg tgg 33 Asp Thr
Tyr Arg Val His Ala Ser Asp Ala Trp 1 5 10 45 11 PRT Artificial
Generated from randomly recombinant DNA part of MAH. 45 Asp Thr Tyr
Arg Val His Ala Ser Asp Ala Trp 1 5 10 46 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 46 gac act tac
ctg gtg cat gtt ccg gat cgg gtt 33 Asp Thr Tyr Leu Val His Val Pro
Asp Arg Val 1 5 10 47 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 47 Asp Thr Tyr Leu Val His Val Pro Asp
Arg Val 1 5 10 48 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 48 gac act tac ctt ggg cat tat cct gat
ctg tgg 33 Asp Thr Tyr Leu Gly His Tyr Pro Asp Leu Trp 1 5 10 49 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
49 Asp Thr Tyr Leu Gly His Tyr Pro Asp Leu Trp 1 5 10 50 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 50
gac act tac cag cct cat gcg ctt gat cgt tgt 33 Asp Thr Tyr Gln Pro
His Ala Leu Asp Arg Cys 1 5 10 51 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 51 Asp Thr Tyr Gln Pro His
Ala Leu Asp Arg Cys 1 5 10 52 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 52 gac act tac cgg gct cat
ttg ggg gat tcg act 33 Asp Thr Tyr Arg Ala His Leu Gly Asp Ser Thr
1 5 10 53 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 53 Asp Thr Tyr Arg Ala His Leu Gly Asp Ser Thr 1 5 10
54 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 54 gac act tac gcg gag cat gct tat gat ttt gcg 33 Asp Thr
Tyr Ala Glu His Ala Tyr Asp Phe Ala 1 5 10 55 11 PRT Artificial
Generated from randomly recombinant DNA part of MAH. 55 Asp Thr Tyr
Ala Glu His Ala Tyr Asp Phe Ala 1 5 10 56 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 56 gac act tac
cgt tat cat gct act gat gct cgg 33 Asp Thr Tyr Arg Tyr His Ala Thr
Asp Ala Arg 1 5 10 57 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 57 Asp Thr Tyr Arg Tyr His Ala Thr Asp
Ala Arg 1 5 10 58 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 58 gac act tac tgt ctt cat tct ctt gat
cgt ctg 33 Asp Thr Tyr Cys Leu His Ser Leu Asp Arg Leu 1 5 10 59 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
59 Asp Thr Tyr Cys Leu His Ser Leu Asp Arg Leu 1 5 10 60 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 60
gac act tac ctg ttt cat tgt tat gat gcg gag 33 Asp Thr Tyr Leu Phe
His Cys Tyr Asp Ala Glu 1 5 10 61 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 61 Asp Thr Tyr Leu Phe His
Cys Tyr Asp Ala Glu 1 5 10 62 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 62 gac act tac atg tcg cat
gat gtt gat cgt tct 33 Asp Thr Tyr Met Ser His Asp Val Asp Arg Ser
1 5 10 63 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 63 Asp Thr Tyr Met Ser His Asp Val Asp Arg Ser 1 5 10
64 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 64 gac act tac gct gct cat tgt ttg gat ggt atg 33 Asp Thr
Tyr Ala Ala His Cys Leu Asp Gly Met 1 5 10 65 11 PRT Artificial
Generated from randomly recombinant DNA part of MAH. 65 Asp Thr Tyr
Ala Ala His Cys Leu Asp Gly Met 1 5 10 66 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 66 gac act tac
tct agt cat tct gtg gat ttg agg 33 Asp Thr Tyr Ser Ser His Ser Val
Asp Leu Arg 1 5 10 67 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 67 Asp Thr Tyr Ser Ser His Ser Val Asp
Leu Arg 1 5 10 68 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 68 gac act tac tgt att cat att ctg gat
cgt cct 33 Asp Thr Tyr Cys Ile His Ile Leu Asp Arg Pro 1 5 10 69 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
69 Asp Thr Tyr Cys Ile His Ile Leu Asp Arg Pro 1 5 10 70 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 70
gac act tac cgt ctt cat gtt agg gat ttt tgg 33 Asp Thr Tyr Arg Leu
His Val Arg Asp Phe Trp 1 5 10 71 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 71 Asp Thr Tyr Arg Leu His
Val Arg Asp Phe Trp 1 5 10 72 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 72 gac act tac gtg tgg cat
tgt gct gat gtt cgg 33 Asp Thr Tyr Val Trp His Cys Ala Asp Val Arg
1 5 10 73 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 73 Asp Thr Tyr Val Trp His Cys Ala Asp Val Arg 1 5 10
74 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 74 gac act tac gcg gat cat ctt gct gat tat gat 33 Asp Thr
Tyr Ala Asp His Leu Ala Asp Tyr Asp 1 5 10 75 11 PRT Artificial
Generated from randomly recombinant DNA part of MAH. 75 Asp Thr Tyr
Ala Asp His Leu Ala Asp Tyr Asp 1 5 10 76 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 76 gac act tac
ggg ctg cat ggt atg gat ggt gct 33 Asp Thr Tyr Gly Leu His Gly Met
Asp Gly Ala 1 5 10 77 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 77 Asp Thr Tyr Gly Leu His Gly Met Asp
Gly Ala 1 5 10 78 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 78 gac act tac ctg ccg cat ggg tct gat
ctt cgt 33 Asp Thr Tyr Leu Pro His Gly Ser Asp Leu Arg 1 5 10 79 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
79 Asp Thr Tyr Leu Pro His Gly Ser Asp Leu Arg 1 5 10 80 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 80
gac act tac ggt cct cat tcg cgt gat cgt agg 33 Asp Thr Tyr Gly Pro
His Ser Arg Asp Arg Arg 1 5 10 81 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 81 Asp Thr Tyr Gly Pro His
Ser Arg Asp Arg Arg 1 5 10 82 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 82 gac act tac acg gtg cat
ccg gct gat tgt gct 33 Asp Thr Tyr Thr Val His Pro Ala Asp Cys Ala
1 5 10 83 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 83 Asp Thr Tyr Thr Val His Pro Ala Asp Cys Ala 1 5 10
84 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 84 gac act tac cat atg cat acg tat gat ttt gtt 33 Asp Thr
Tyr His Met His Thr Tyr Asp Phe Val 1 5 10 85 11 PRT Artificial
Generated from randomly recombinant DNA part of MAH. 85 Asp Thr Tyr
His Met His Thr Tyr Asp Phe Val 1 5 10 86 33 DNA Artificial
Generated from randomly recombinant DNA part of MAH. 86 gac act tac
gtt cgt cat agt tgt gat cgg gcg 33 Asp Thr Tyr Val Arg His Ser Cys
Asp Arg Ala 1 5 10 87 11 PRT Artificial Generated from randomly
recombinant DNA part of MAH. 87 Asp Thr Tyr Val Arg His Ser Cys Asp
Arg Ala 1 5 10 88 33 DNA Artificial Generated from randomly
recombinant DNA part of MAH. 88 gac act tac gtg tgg cat ggg cgg gat
ggt gct 33 Asp Thr Tyr Val Trp His Gly Arg Asp Gly Ala 1 5 10 89 11
PRT Artificial Generated from randomly recombinant DNA part of MAH.
89 Asp Thr Tyr Val Trp His Gly Arg Asp Gly Ala 1 5 10 90 33 DNA
Artificial Generated from randomly recombinant DNA part of MAH. 90
gac act tac gcg atg cat gcg ttg gat gtt ggt 33 Asp Thr Tyr Ala Met
His Ala Leu Asp Val Gly 1 5 10 91 11 PRT Artificial Generated from
randomly recombinant DNA part of MAH. 91 Asp Thr Tyr Ala Met His
Ala Leu Asp Val Gly 1 5 10 92 33 DNA Artificial Generated from
randomly recombinant DNA part of MAH. 92 gac act tac agt tcg cat
tgg ggt gat ccg tgg 33 Asp Thr Tyr Ser Ser His Trp Gly Asp Pro Trp
1 5 10 93 11 PRT Artificial Generated from randomly recombinant DNA
part of MAH. 93 Asp Thr Tyr Ser Ser His Trp Gly Asp Pro Trp 1 5 10
94 33 DNA Artificial Generated from randomly recombinant DNA part
of MAH. 94 gac act tac att ggt cat acg gct gat ctt ttt
33 Asp Thr Tyr Ile Gly His Thr Ala Asp Leu Phe 1 5 10 95 11 PRT
Artificial Generated from randomly recombinant DNA part of MAH. 95
Asp Thr Tyr Ile Gly His Thr Ala Asp Leu Phe 1 5 10
* * * * *